Nitrogen Excretion in Developing Chick Embryos
by JAMES R. FISHER 1 and ROBERT E. EAKIN
From the Biochemical Institute and the Department of Chemistry,
The University of Texas, Austin
INTRODUCTION
O F the many examples of metabolic changes occurring during development, the
most widely cited is that in developing chicks wherein the end product of nitrogen metabolism has been assumed to shift from ammonia to urea and finally to
uric acid (Needham, 1931). This system appeared to be ideal for a proposed
study of the mechanisms bringing about such changes in metabolism, providing
the occurrence of these changes could be further substantiated. The experimental
evidence upon which this pattern had been postulated consisted entirely of
measurements of the total amounts of ammonia, urea, and uric acid in the allantois and the ratio of their total weights to the weight of the embryo. Nothing was
reported concerning the changes in concentration of these substances nor their
presence outside the allantois. Lack of this information makes it impossible to
conclude that net synthesis occurs, particularly in the cases of ammonia and
urea where the total amounts present are quite small.
If such a series of changes occurs in the excretion pattern of the developing
chick, it is necessary that the enzymatic patterns of the organism undergo a concurrent series of transformations. In this connexion there appears in the literature
only an incomplete analysis of arginase activity in developing chicks (Needham
&Brachet, 1935).
In this paper there is presented an analysis of the nitrogenous contents of the
allantois and the entire egg, a study of the fates of injected nitrogenous substances, and an analysis of embryos for the enzymes urease and arginase. These
investigations have yielded evidence which we believe clearly shows that ammonia is not an excretion product and that the embryo does not pass through stages
of ammonia or urea excretion characteristic of its evolutionary ancestry.
MATERIALS AND METHODS
Eggs used for these studies came from a flock of white leghorns and were
incubated at 39° C. in a commercial incubator. Eggs were injected by placing
1
Rosalie B. Hite Postdoctoral Fellow. Present address: Chemistry Department, Florida State
University, Tallahassee, Florida, U.S.A.
[J. Embryol. exp. Morph. Vol. 5, Part 3, pp. 215-224, September 1957]
5545.5
O
216
J. R. FISHER AND R. E. EAKIN
0-5 ml. of either distilled water or the appropriate solution in the air-sac. Allantoic fluid was removed with a hypodermic syringe through a hole in the blunt
end of the egg.
Chemical determinations
Ammonia was determined by the method of Boyce (1950) substituting DowCorning antifoam A for the antifoam agent used in the original procedure. This
method involves the removal of ammonia from complex mixtures by passing air
through an alkaline aliquot into 0 1 N H2SO4 which traps the volatile ammonia.
The quantity present is then determined by the use of Nesaler's Reagent according to the method of Folin & Wu (1919). All analyses were run in duplicate.
Urea was determined by treating the sample with a standard solution of jack
bean meal urease (Folin & Wu, 1919) and measuring the resulting ammonia by
the method described above.
Uric acid in the allantoic fluid was determined by the glycerol-silicate method
ofForshameffl/.(1948).
Total nitrogen was determined by a modified Kjeldahl digestion (Ma &
Zuazaga, 1942) followed by nesalerization according to the procedure used for
ammonia determinations.
Enzyme determinations
Urease was measured by the method of Van Slyke & Cullen (1914) wherein a
tissue homogenate is incubated with a standard buffered (pH 7-0) solution of
urea, at room temperature for various time intervals. After stopping the enzymatic activity by addition of acid the amount of ammonia formed is determined
as described above.
Arginase activity was determined according to the procedure of Van Slyke
& Archibald (1946) as follows: (1) tissues are homogenized in an appropriate
amount of distilled water; (2) 0-25 ml. of a 20 per cent, solution of MnCl2.4H2O
is added to a 5 ml. aliquot of the homogenate; (3) the mixture is incubated for 20
minutes at 56° C. (to activate the enzyme); (4) after cooling, 2 5 ml. of an 18 per
cent, solution of arginine (adjusted to pH 9-5) is added; (5) the reaction mixture
is incubated at room temperature (approximately 25° C); and (6) 1-ml. samples
are removed after 0,20,40, and 60 minutes of incubation and placed in test-tubes
maintained for 10 minutes at 90° C. in a water-bath to stop enzymatic activity.
The 1-ml. samples are adjusted to pH 7 with 0-2 M phosphate buffer; the urea
formed is hydrolysed with urease; the resulting ammonia is determined as
described above.
RESULTS AND DISCUSSION
Ammonia excretion
Needham (1926a, 1931) has published data showing that the total amount of
ammonia in the allantois increases during chick development from 3 //g. at 4
days to 56 at 13 days, and has shown that the ratio of allantoic ammonia to
NITROGEN EXCRETION IN CHICK EMBRYOS
217
weight of embryo decreases during this entire period. From these facts he postulated that chicks excrete ammonia in early stages and gradually lose this capacity
with urea becoming the predominant excretory product.
TABLE 1
Ammonia in allantoic fluid and entire egg contents during development
Incubation time
(days)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
Concentration of NH3 Total NHS content
(pg
(pg- f
Allantois Entire egg Allantois Entire egg
8(3)t
11 (5)
10(5)
12(0
8(1)*
8(5)
8(4)
6(3)
6(3)
10(1)
10(5)
11(3)
10(2)
8(2)
8(3)
9(3)
8(3)
7(4)
9(0
12(2)
7(2)
3
10
19
21
28
55
64
350
500
450
550
500
450
350
350
400
350
300
400
550
300
* Number of pooled samples analysed separately and values averaged,
t Number of eggs analysed separately and values averaged.
Our data are in agreement with his experimental findings that the total amount
of ammonia in the allantois increases during development; however, measurements (Table 1) show that the concentration remains constant. The concentration
of ammonia in the whole egg contents (homogenized) was found to equal that in
the allantois, and the total amount in the developing egg remained relatively
constant from the 1st through the 13th day (Table 1). Samples were pooled
from a number of allantoic sacs (e.g. 13 on 5th day, 3 on 11th day) to obtain
adequate volumes for analysis. The volumes of allantoic sacs were determined
from a standard plot made by removing the fluid contents with a syringe from
ten replicate eggs at each day and measuring the volume obtained. These values
agreed very closely with those found by Fiske & Boyden (1926).
When 1 mg. of ammonia nitrogen (as a buffered ammonium sulphate solution)
is placed in the air-sac of eggs which have been incubated for 6 days, it becomes
equally distributed between the yolk and allantois raising the concentration of
ammonia in each threefold. This excess ammonia disappears rapidly, the value
for both yolk and allantois becoming equal to the controls injected with distilled
water within 2 days (Table 2).
If the early chick embryo, from the 1st to the 5th day, is an ammonia-excreting
organism, it is necessary that urease be present, otherwise purine nitrogen would
218
J. R. F I S H E R A N D R. E. E A K I N
not be eliminated as ammonia. All attempts to demonstrate urease activity have
been unsuccessful, even in the blastodiscs of unincubated eggs. The methods
used were so sensitive that the production of 1 /*g. of ammonia nitrogen by an
entire embryo in 1 hour would have been detected.
TABLE 2
Fate of ammonia injected as ammonium sulphate (1 0 mg. N) into 6-day-old
developing eggs
Concentration NH3
(fig. Njml.)
Incubation time
(days)
Allantois
NH3 injected
6
6-2
6-4
6-6
8
9
10
11
31 (2)*
23(1)
10(5)
7(2)
8(4)
10(3)
8(7)
Yolk
> injected
8(5)*
11 (2)
9(1)
7(2)
6(3)
6(3)
10(1)
10(5)
NH3 injected
H tO injected
7(0*
29(1)*
17(1)
21 (4)
8(3)
9(2)
10(1)
8(1)
14(1)
12(1)
6(0
* Number of pooled samples analysed separately and values averaged.
These data show that (1) the total amount of ammonia in the allantois increases
during development; (2) the concentration of ammonia in the allantois remains
constant from the 5th through at least the 11th day; (3) throughout this period
the concentration of ammonia outside the allantois equals that inside; (4) ammonia injected as ammonium sulphate into a developing egg rapidly becomes distributed uniformly in the yolk and allantois, increasing the concentrations in each
to the same extent; (5) the excess ammonia resulting from the injection disappears
from both the allantois and the yolk in less than 2 days' time; and (6) no urease
activity is detectable from as early as the blastoderm stage.
The existing evidence does not warrant any conclusion concerning the presence of an active ammonia excreting mechanism during the first 5 days of
development, and that after the 5th day there is no net excretion of ammonia
into the allantoic sac.
Urea excretion
As in the case of ammonia, Needham (19266,1931) has reported data showing
(1) that the total amount of urea in the allantois increases during chick development; (2) that the ratio of urea to the weight of the embryo increases from the 4th
to the 8th day; and (3) that this ratio decreases from the 8th day to the time of
hatching. He has postulated from these facts that the chick passes through a stage
of high urea excretion.
N I T R O G E N E X C R E T I O N IN C H I C K E M B R Y O S
219
Our analyses are in agreement that the total amount of urea in the allantois
increases during development (Table 3). It was also found (1) that, unlike ammonia, the allantoic concentration of urea does not remain constant but increases
from the 5th through at least the 11th day; (2) that the total amount of urea in
the entire egg contents increases during the entire period studied; and (3) that
the concentration of urea in the allantoic fluid is several fold higher than in the
surrounding fluids (by the 11th day the allantois contains one-half of the urea
present inside the entire egg, see Table 3).
TABLE 3
Urea in allantoic fluid and in entire egg contents during development
Incubation time
(days)
Concentration of urea
(fig. N/ml.)
Allantois Entire egg
6
7
8
9
10
11
(l*g- N)
Allantois
2(Dt
0
1
2
3
4
5
Total urea content
3(1)
4(3)
4(1)
5(3)
10(0*
11(3)
16(2)
21 (3)
26(3)
29(1)
31(5)
3(0
8(1)
6(1)
6(1)
12(2)
9(2)
2
14
37
69
112
154
198
Entire egg
100
150
200
200
200
150
350
250
250
550
400
* Number of pooled samples analysed separately and values averaged,
t Number of eggs analysed separately and values averaged.
TABLE 4
Fate of urea (10 htg. N) injected into 6-day-old developing eggs
Concentration of urea
in the allantois
dig. N/ml.)
Incubation time
(days)
Urea
injected
10(1)*
11(3)
5
6
6-2
7
8
9
10
11
Water
injected
Total amount of
urea n the
allantois (fig. N)
Water
Urea
injected
injected
50(1)*
84(1)
77(1)
94(1)
93(1)
92(1)
16(2)
21(3)
26(3)
29(1)
31(5)
2
14
70
190
250
400
490
580
37
69
112
154
198
Number of pooled samples analysed separately and values averaged.
220
J. R. FISHER AND R. E. EAKIN
When 1 mg. of urea nitrogen (as a urea solution) is placed in the air-sacs of
eggs, which have been incubated for 6 days, it rapidly becomes concentrated in
the allantoic sac and remains there (Table 4). By the 1 lth day 40 per cent, of all
the injected urea has been concentrated in the allantois even though the sac
occupies only 10 per cent, of the total volume.
If the developing chick embryo passes through a stage wherein urea is the
primary excretion product and shifts to a stage wherein uric acid is the primary
product, one should expect to find a shift in the relative amounts of each being
excreted. The daily increases in concentration of urea in the allantoic fluid from
the earliest time measured (5th day) through the 1 lth were approximately 1 (?),
5, 5,5, 3, and 2 /*g. of urea N/ml. (see Table 3); comparable values for uric acid
were 30,36,32,46,28, and 31 jxg. uric acid N/ml. (see Table 6). Hence, from the
earliest times at which measurement could be made the relative amounts of each
excreted were reasonably constant and uric acid is the major excretory product
as early as the 5th day.
The presence of a ureotelic metabolism in a developing chick would require
that the enzyme arginase be present in order for protein nitrogen to be eliminated. It has been shown (Needham & Brachet, 1935) that the arginase activity
per milligramme moist weight of developing chick embryos decreases during
development from the 3rd to the 15th day. If the arginase in the early chick
embryo is the result of a recapitulation of a ureotelic excretory metabolism
certain organs (primarily the liver) should possess a higher concentration of the
enzyme than others (Hunter & Dauphinee, 1924).
TABLE 5
Arginase activity in developing chick embryos
Arginase activity*
ing. arginine hydrolysed\hour\mg. moist tissue)
Incubation time
(days)
Entire
embryo
2-5
30
40
50
60
7-0
80
90
100
110
120
130
140
150
400
310
140
10-5
8-5
7-0
60
5-5
4-5
30
2-5
20
1-5
10
Head of
embryo
Body of
embryo
Liver of
embryo
7-8
6-9
9-7
3-4
4-3
3-3
21
1-8
0-9
1-9
10
* Each value reported is the average of at least two separate determinations on
pooled samples from several embryos.
NITROGEN EXCRETION IN CHICK EMBRYOS
221
We have measured the arginase activity of intact embryos from 2-5 to 15 days,
of separated heads and bodies from the 7th to the 15th day, and of isolated livers
of 7-day-old chick embryos. No differences in activity were found (Table 5). On
the basis of these data it is concluded that during the mesonephric stage of
development chicks do not recapitulate the arginase distribution associated with
a typical ureotelic physiology.
These studies show: (1) that the total amount of urea in the allantois increases
during development; (2) that the concentration of urea in the allantoic sac also
increases from the 5th through at least the 11th day; (3) that the concentration
of urea is greater inside the allantois than outside; (4) that the injected urea
becomes concentrated in the allantois; (5) the fraction of nitrogen excreted as
urea remains relatively constant from the 6th through at least the 1 lth day; and
(6) that arginase is not distributed among the various tissues of the developing
chick in a manner characteristic of ureotelic organisms.
It is concluded that the developing chick excretes urea, but that the rate of
urea excretion does not reach a maximum at any particular stage, and that the
organism does not recapitulate a typical ureotelic excretion physiology.
Uric acid excretion
Although uric acid excretion has been extensively studied by many investigators (a list has been compiled by Needham, 1931), it was included in our studies
for purposes of obtaining a more complete picture (Table 6).
TABLE 6
Fate of uric acid (0 5 mg. N) injected into 6-day-old developing eggs
Concentration of uric
acid in the allantois
(tig. Nlml.)
Incubation time
(days)
5
6
6-2
6-4
6-6
7
8
9
10
11
Uric acid
injected
Water
injected
74(4)*
81(2)
99(2)
90(5)
117(2)
185 (2)
247 (2)
295 (1)
37(4)
45(4)
52(2)
54(4)
73(5)
105 (2)
151 (2)
179 (3)
210(1)
Total amount of uric
acid in the allantois
fag-N)
Uric acid
injected
7(0*
104
130
180
210
390
790
1,300
1,860
Water
injected
21
48
65
86
105
168
340
650
950
1,320
* Number of pooled samples analysed separately and values averaged.
When half a milligramme of uric acid nitrogen (as a sodium ureate solution
adjusted to pH 7) was placed in the air-sac of eggs which have been incubated
222
J. R. FISHER AND R. E. EAKIN
for 6 days, it gradually accumulated in the allantoic sac until essentially all of it
appeared there by the 1 lth day (Table 6).
By dividing the amount of uric acid by the weight of the embryo, Needham
(19266,1931) obtained an excretion curve which is essentially zero from the 4th
to the 7th day, which raises to a peak at the 11th day and then decreases. This
curve can be interpreted as meaning that the embryo changes from a ureaexcreting organism to an essentially uric-acid-excreting organism during the
period from the 7th to the 11th day. If the change were as dramatic as would
appear from this particular method of analysis of the uric-acid data, one should
expect to find a shift in the relative amounts of each being excreted. As was
shown previously (section on urea excretion) the daily increments in concentration in the allantois due to urea and uric acid are essentially constant from the
5th through at least the 11th day, and throughout this entire period the value
for urea is only about one-sixth that for uric acid. Hence, from the earliest times
at which measurements could be made the relative amounts of each excreted
were reasonably constant and it is concluded that uric acid is the major excretory
product as early as the 5th day.
Nitrogenous composition of allantoic fluid in developing chicks
To conclude this study of nitrogen excretion in developing chicks the total
nitrogen in the allantois was determined to find out how much of the nitrogen
present was accounted for by ammonia, urea, and uric acid.
Our analyses leave about 60 per cent, of the nitrogen to be accounted for. This
is considerably more than calculated by Needham (1931) from data compiled
from several investigators. Our unassignable fraction is larger than his value
primarily because our uric-acid values were lower than those he used (Fiske &
Boyden, 1926); however, our values are actually higher than Needham's (1926)
own experimental values.
TABLE 7
Nitrogenous contents of the allantoic sacs of developing chicks
Per cent, of total nitrogen
Incubation time
(days)
Total nitrogen
(tig. N/sac)
NH 3
Urea
Uric acid
5
6
7
8
9
10
11
93
325
620
1,060
1,980
2,900
3,700
3-2
3-1
31
20
1-4
1-9
1-7
2-2
4-3
60
6-5
5-7
5-3
5-4
2-2
17-5
290
321
32-8
32-4
35-7
N I T R O G E N E X C R E T I O N IN C H I C K E M B R Y O S
223
CONCLUSIONS
Ammonia excretion
Because (1) the concentration of ammonia inside and outside the allantois are
constant and equal from the 5th through at least the 11th day; (2) ammonia
injected into the egg moves rapidly and equally into the yolk and allantois
(rapidly disappearing from both); and (3) no urease activity was detected, it is
concluded that ammonia is not an excretion product after the 5th day of development, that it need not necessarily be considered a product of active excretion
prior to this time, and that the increase in total ammonia in the allantois is due
only to the increasing volume of that organ.
Urea excretion
Because (1) the concentration of urea inside the allantois is greater than that
outside and increases during development; (2) urea injected into the egg moves
rapidly into the allantois and remains there; (3) the increments of urea concentration in the allantois are essentially constant from the 5th to the 11th day; and
(4) arginase is uniformly distributed among the various tissues of the embryo, it
is concluded that although urea is an excretion product from the 5 th through
at least the 11th day, no predominantly ureotelic stage exists during chick
development.
Uric acid excretion
Because (1) the concentration and total amount of uric acid in the allantois
increases from the 5th to at least the 1 lth day; (2) uric acid injected into the egg
accumulates in the allantois; and (3) the daily increments of uric-acid concentration in the allantois are equal from the 5th through at least the 11th day, it is
concluded that uric acid is a major excretion product from the 6th to the 1 lth day
and that no marked shift from urea to uric-acid excretion occurs during this
period.
Total nitrogen in the allantois
The total amount of nitrogen was determined and 40 per cent, was accounted
for as ammonia, urea, or uric acid.
SUMMARY
1. An analysis, more extensive than hitherto, has been made of nitrogen excretion in developing chicks. It was found that the concentration of ammonia in the
allantoic fluid was constant from the 5th to the 11th day and equalled the concentration in the extra-allantoic fluids, e.g. yolk. The increase into total ammonia
content of the allantois during development is apparently due to the increase in
volume of the organ and not to excretion by the embryo.
224
J. R. FISHER AND R. E. EAKIN
2. The concentration of urea in the allantoic fluid increases during development and injected urea concentrates in that organ.
3. The total amount and concentration of uric acid in allantoic fluid increases
during development. The ratio of urea nitrogen to uric-acid nitrogen is relatively
constant from the 6th to the 11th day of development indicating that no major
shift from urea excretion to uric-acid excretion occurs.
4. No urease activity was observed even in the blastodiscs of unincubated
eggs.
5. The distribution of arginase activity in 7-day-old chicks did not resemble
that of ureotelic organisms. Activity of heads, bodies, and livers were essentially
the same.
6. From these results it was concluded that ammonia is not an excretion product and that the developing chick does not pass through either a typical ammonotelic or ureotelic stage of excretion.
REFERENCES
BOYCE, E. B. (1950). Report on free ammonia in eggs. / . Ass. off. agric. Chem. 33, 703-7.
FISKE, C. H., & BOYDEN, E. A. (1926). Nitrogen metabolism in the chick embryo. / . biol. Chem.
70, 535-56.
FOLIN, O'., & Wu, H. (1919). A system of blood analysis. /. biol. Chem. 38, 81-110.
FORSHAM, P. H., THORN, G. W., PRUNTY, F. T. G., & HILLS, A. G. (1948). Clinical studies with
pituitary adrenocorticotropin. / . clin. Endocrin. 8, 15-66.
HUNTER, A., & DAUPHINEE, J. A. (1924). Quantitative studies concerning the distribution of
arginase in fishes and other animals. Proc. roy. Soc. B 97, 227-42.
MA, T. S., & ZUAZAGA, G. (1942). Micro-Kjeldahl determination of nitrogen. Industr. engng.
Chem. Anal., Ed. 14, 280-2.
NEEDHAM, J. (1926a). The ammonia content of the developing avian egg and the theory of recapitulation. Brit. J. exp. Biol. 4, 145-54.
(19266). The uric acid content and the general protein metabolism of the developing avian
egg. Brit. J. exp. Biol. 4, 114-44.
(1931). Chemical Embryology. Vol.2. Cambridge University Press.
& BRACHET, J. (1935). L'activite* de l'arginase pendant le developpement de 1'embryon du
poulet. C.R. Soc. Biol. Paris, 118, 840-2.
VAN SLYKE, D. D., & ARCHIBALD, R. M. (1946). Gasometric and photometric measurement of
arginase activity. / . biol. Chem. 165, 293-309.
& CULLEN, G. E. (1914). A permanent preparation of urease and its use in the determination
of urea. / . biol. Chem. 19, 211-28.
(Manuscript received 26: vii: 56)